Copyright2010, IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY. All rights reserved.

225
ORIGINAL ARTICLE
Iran J Allergy Asthma Immunol
December 2010; 9(4): 225-230.


Effect of Treatment with Intranasal Corticosteroid and Oral Antihistamine on
Cytokine Profiles of Peripheral Blood Mononuclear Cells of Patients with
Allergic Rhinitis Sensitive to Chenopodium album

Shokrollah Farrokhi
1,2
, Tahereh Mousavi
1,3,4
, Saba Arshi
5
, Naser Javahertarash
5
, Abdolreza Varasteh
6
,
Reza Falak
6
, Nima Rezaei
7
, Alireza Salekmoghadam
1,4


1
Department of Immunology, Tehran University of Medical Sciences, Tehran, Iran
2
Department of Immunology, Bushehr University of Medical Sciences, Bushehr, Iran
3
Microbial Resistance Research Center, Tehran University of Medical Sciences, Tehran, Iran
4
Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran
5
Department of Allergy

&

Clinical

Immunology,

Rasoul Akram

Hospital,

Tehran

University of

Medical

Sciences,

Tehran,

Iran
6
Immunobiochemistry Lab, Immunology Research Center, Avicenna Research Institute,
Mashhad University of Medical Sciences, Mashhad, Iran
7
Molecular Immunology Research Center; and Department of Immunology, School of Medicine,
Tehran University of Medical Sciences, Tehran, Iran

Received: 20 April 2009 ; Received in revised form: 12 June 2009 ; Accepted: 1 November 2009


ABSTRACT

Patients with allergic rhinitis (AR) show increased production of the Th2-related cytokines. Almost
always, intranasal corticosteroid (INC) and antihistamine are used as routine therapy of AR. This study
was performed to determine the in vitro secretion of cytokines profiles of PBMCs in patients with AR
sensitive to Chenopodium album (Ch.a) pollens before and after treatment with INC (Fluticasone
propionate) and oral antihistamine (Loratadine).
PBMCs of 20 patients with AR, were tested in vitro for cytokine production. These cells were
stimulated with natural or recombinant Ch.a. The levels of IL-4, IL-13 and IFN-, were measured in
supernatants of cultured cell 96h after stimulation using ELISA. The PBMCs of 20 normal individuals
were also similarly treared for comparison of results. The production of IL-4 by the patients cells
stimulated with either Ch.a or rCh.a was significantly higher than normal levels before therapy (p=0.04
and p=0.02, respectively). After therapy, a significant decrease in production of IL-4 and a significant
increase in production of IL-10 were found in PBMCs stimulated with natural Ch.a, in comparison to
the results before stimulation (p=0.03 for IL-4; p=0.04 for IL-10). Similarly, these results were seen in
the production of IL-4 and IL-10 stimulated with rCh.a allergen after therapy in comparison to the
results before stimulation (p=0.01 for IL-4; p=0.03 for IL-10).
This study suggests INC (Fluticasone propionate) and oral antihistamine (Loratadine) have the
capacity to inhibit the production of IL-4 and shift Th2/Th1 responses, probably due to increase the
level of immunoregulatory IL-10. Therefore, it could be concluded that therapy with INC and
antihistamine has pharmacologic and immunologic therapeutic effects on AR patients.

Key words: Allergic Rhinitis; Antihistamine; Cytokines; ELISA; Intranasal Corticosteroid; PBMCs

Corresponding Author: Tahereh Mousavi, PhD;
Department of Immunology, Medical College, Iran University of

Medical Sciences, Tehran, Iran. Tel/Fax: (+98 21) 8805 8652,
E-mail: yasaha@iums.ac.ir

Sh. Farrokhi, et al.
226/ IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 9, No. 4, December 2010
INTRODUCTION

Allergic rhinitis (AR) is a chronic IgE-mediated
inflammatory disease, which is triggered by allergens,
including seasonal allergens such as weeds, pollen. The
prevalence of AR is growing up worldwide and it is
estimated that up to 20% of general population are
affected by AR.
1
Chenopodium album (Ch.a) is one of
the most allergenic plants with high abundance growth
in most parts of Iran.
2
At present, Ch.a natural extracts
are used for diagnosis and immunotherapy of AR
patients. The natural homemade extract of Ch.a has
been prepared and examined in skin prick test in a
previous study in Iran.
3

Also, Mousavi et al induced asthma in animal
model using homemade natural extract of Ch.a
which resulted in increasing the levels of IL-4 and IL-5
as well as specific Ch.a IgE in mice.
4
It has been
suggested that increased activity of T-helper (Th)2
cells, which produce IL-4, IL-13 and IL-5 cytokines
contribute to allergic inflammation.
5,6
Intranasal
corticosteroid (INC) and antihistamine are introduced
as the first line of therapy for AR by the Allergic
Rhinitis and its Impact on Asthma (ARIA) global
guidelines.
7

Several studies showed that INC reduced the level
of cytokines secreted by Th2 cells and inhibited levels
of other cytokines such as IL-1, IL-8, GM-CSF and
TNF- in AR.
8-12
These reports indicate that
maintenance treatment with INC can have anti-
inflammatory nasal effects.
The main goal of this project was, without
intervention in the usual treatment of the patients with
AR, to study the effects of intranasal corticosteroid and
oral antihistamine on cytokine production of PBMCs of
patients with AR.
Moreover, we studied cytokine profiles of Th1, Th2
and immunoregulatory cytokines after therapy of AR
patients without in vivo intervention.
For this, we explored the effects of combination use
of INC (Fluticasone propionate) and antihistamine
(Loratadine) on the Th1 cytokine [with assay of
Interferon (IFN)-], Th2 cytokines (with assay of IL-4
and IL-13) and immunoregulatory cytokine (with assay
of IL-10) profiles secreted by peripheral blood
mononuclear cells (PBMCs) isolated from patients with
AR and from normal individuals exposed to natural or
recombinant Ch.a (rCh.a) allergens in vitro.

MATERIALS AND METHODS

Subjects
Twenty patients with AR were enrolled in this
study. Demographic data of all subjects are shown in
table 1. The inclusion criteria were subjects with age
range of 18 years, presence o f rhinitis symptoms,
history of rhinitis 1 year, and positive SPT to Ch.a
allergen (Stallergen, France).
13
We excluded subjects
with more than 50 years who did not participate in
second time sampling, and had high titer of total IgE
due to nonallergic causes and 20 normal individuals
with no history of AR and a negative skin prick test
(SPT) to the Ch.a allergen. The patients were classified
according to the ARIA classification.
7
All participants
had been free of medications 3 months prior to the
study. INC (Fluticasone propionate, 2 puff, morning,
gsk, UK) and antihistamine (Loratadine, 10 mg, orally,
daily, Razi co, Iran) had been prescribed for all patients
as routine therapy. Blood samples were collected
before starting therapy at first visits and 6 weeks later
during pollination season of Ch.a (late summer and
fall). The study was approved by the local ethics
committee. Written informed consents were also
obtained from all subjects.

Study Design
Five milliliters (ml) of venipuncture blood from all
subjects were collected into falcon tubes containing
EDTA (EthyleneDiamineTetraacetic Acid). PBMCs
were immediately isolated using Ficoll-Paque
(Fresenius Kobi Noge As for Axis-Shield Poc As, Oslo,
Norway) and gradient centrifugation. Isolated PBMCs
were cultured in a 24-well plate (J ET Biofil, Canada)
in RPMI 1640 (Biosera, UK) containing 10% fetal calf
serum (FCS) (Biosera, UK), 50 g/ml penicillin, 50
g/ml streptomycin (Biosera, UK), 0.3 mg/mL L-
glutamine (Biosera, UK), at a density of 1106 cells
per well and incubated (at 37C and in 5% CO2) in the
presence of 50 g/ml natural Ch.a or 10 g/ml rCh.a,
separately. Supernatants were collected before and after
therapy of patients and aforementioned cytokines were
measured.
Natural Ch.a allergen was extracted and purified
from whole Ch.a pollen as described before.
3
rCh.a
allergen was prepared in Immunobiochemistry Lab,
Immunology Research Center, Avicenna Research
Institute, Mashhad University of Medical Sciences,
Mashhad, Iran.

Effect of Intranasal Corticosteroid and Oral Antihistamine Treatment on Allergic Rhinitis
Vol. 9, No. 4, December 2010 IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /227
Table 1. Subjects Characteristics
Features Allergic rhinitis
patients
Normal control
Number (n) 20 20
Age (yr) 296 298
Gender 7 M, 13 F 7 M, 13 F
SPT (mmof Weal) 7.73.2 0
Total IgE (IU/ml) 234.7151.15 47.512.6
ARIA classification:
Mild intermittent 1 -
Moderate- severe intermittent 0 -
Mild persistent 9 -
Moderate- severe persistent 10 -
Skin Prick Test (SPT), Allergic Rhinitis and Its Impact on Asthma (ARIA)


Levels of IFN-, IL-4, IL-10, and IL-13 were
measured in supernatants 96 h after culture, using
ELISA sets from U-CyTech biosciences (Utrecht, The
Netherlands) with sensitivity of of 2 pg/ml. Total IgE
were also measured with ELISA kit in sera of all AR
patients with sensitivity 1 IU/l (PADTAN ELM, Iran).

Statistics
Statistical analysis was performed using SPSS 16
software package (San Diego, CA). Comparisons of the
results before and after treatments were also done using
Wilcoxon Signed Ranks Test. P <0.05 were considered
statistically significant.

RESULTS

As shown in table 1, there was no significant
difference between these two groups with respect to
gender distribution and age. Compared with the
patients, normal subjects were negative for allergen
skin test and did not show any clinical symptoms of AR
at enrolment.

Effect of INC and Antihistamine on IFN-
Production
The levels of secreted of IFN- by natural Ch.a or
rCh.a-stimulated PBMCs from patients before and after
treatments and normal individuals are shown in table 2.
There was not any significant difference on IFN-
production before therapy of patients compared to the
baselines obtained from normal individuals. There was
also no significant difference on IFN- production in
patients before and after therapy.

Effect of INC and Antihistamine on IL-4 and IL-13
Production
PBMCs of AR patients with moderate- severe
persistent AR (10 cases) secreted significantly higher
IL-4 in comparison with PBMCs of other AR patients
(10 cases) (P=0.04).
To investigate the inhibitory effects of INC and
antihistamineon, the cytokine secreted in response to
Ch.a allergens by PBMCs, including IL-4 and IL-13 in
supernatants were measured. Stimulated PBMCs from
patients with AR secreted significantly higher IL-4 in
supernatants after exposure to natural Ch.a or rCh.a,
compared to normal group (P=0.04 and P=0.02,
respectively), whereas after therapy of AR patients, the
level of IL-4 in supernatant of their PBMC decreased
significantly. (P=0.03 and P=0.01, respectively).

Effect of INC and Antihistamine on IL-10
(Immunoregulatory Cytokine) Production
In order to demonstrate whether IL-10 could be an
immunoregulatory cytokine to inhibit Th2 responses,
we measured IL-10 and found that secreted IL-10 in
supernatants after therapy of patients was significantly
increased, after exposure to natural Ch.a or rCh.a
allergens (P=0.04 and P=0.03 for both allergens,
respectively) (Table 2).
Sh. Farrokhi, et al.
228/ IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 9, No. 4, December 2010
Table 2. Cytokine profiles of natural or recombinant Ch.a (rCh.a) stimulated PBMCs from patients with AR before and
after treatments in comparison with stimulated PBMCs from normal control.
Cytokine
(pg/ml)
PBMCs exposed to natural Ch.a PBMCs exposed to rCh.a
AR patients
Normal
controls
P value
AR patients
Normal
controls
P value
Before
treatment
After
treatment


Before
treatment
After
treatment
IFN- 12.75.3 17.73.5 20655.3 0.2 12.35.6 17.32 207.356.1 0.3
IL-4 14.64.8 2.70.2 2.20.6 0.03* 15.55.6 2.20.5 2.60.5 0.01*
IL-13 122.2 11.23 2.80.5 0.7 122 11.33.8 2.40.5 0.9
IL-10 13.34.5 26.72.2 14.44 0.04* 14.14 332.9 7.10.7 0.03*
Chenopodiumalbum(Ch.a), Peripheral Blood Mononuclear Cells (PBMCs), Allergic Rhinitis (AR)
Values are mean standard deviation (SD)
*P<0.05 compared with normal control group

DISCUSSION

Although INC and antihistamine are commonly
used in the treatment of AR, at present the
immunomodulatory effects of these drugs on immune
responses remain unclear.
Allergic reaction inducing AR symptoms can occur
in early and late phases. The late phase reaction occurs
6 to 24 hrs after exposure to an allergen, which is
associated with cellular infiltration of the nasal mucosa
with activated CD4+ Th2 cells and eosinophils.
14

Recent studies showed that cytokines IL-4, IL-5, IL-9
and IL-13 play a crucial role in this inflammatory
reaction,
15,16
while IFN- and IL-10 have been
concerned as cytokines which counter and regulate the
Th2 response by induction of tolerance and protect
from the development of allergic disorders.
17,18
Rudin
et al.
19
indicated that in vitro stimulation of PBMCs
with allergen showed an increased expression of the
Th2-associated cytokines (IL-4, IL-5, IL-9, and IL-13),
whereas recent study by Heaton et al.
20
showed that
PBMCs from both normal and allergic patients to house
dust mites secreted comparable IFN- production after
exposure to a mite extract. Our data are in agreement
with both studies indicating that PBMCs from patients
with AR produced higher IL-4 production.
Previous studies have shown that INC treatment
results in ablation of the late phase allergic reaction,
possibly due to the selective inhibition of cytokines
such as IL-4 and IL-5 expression.
21-23
Ghaffar et al.
24

found that 6-weeks topical gluccocorticosteroid
treatment in subjects with allergic rhinitis induced a
significant reduction in the number of IL-13-expressing
cells. Similarly, our data indicated that the production
of IL-4 by PBMCs of AR patients was significantly
decreased. But IL-10 in supernatants was increased
after therapy with INC and antihistamines. According
to Wolk et al.
25
who indicate that IL-10 is required for
antigen presentation to regulate the nature and extent of
the T helper response, we concluded that increase
production of IL-10 induced by INC and antihistamine
may could regulate the expression of IL-4 by PBMCs.
Sim et al.
26
also showed that treatment of patients with
topical decongestants, as vasoconstrictor, selectively
induced decrease of IL-5. This study confirms
synergistically effect of combination therapy of INC
and antihistamine in patients with AR which enhanced
the production of IL-10. We suggest that these findings
were the effects of both INC and antihistamine on
cytokine profiles of PBMCs of AR patients, because of
baseline effects of them and our assessment of patients
that did not use other drugs.
Altogether, it could be suggested that both natural
Ch.a and rCh.a induce IL-4 as a main Th2-associated
cytokine. Also, INC and antihistamine due to their anti-
inflammatory effects can significantly inhibit the
production of IL-4 which is mediated by
downregulatory effect of IL-10.
One goal of INCs and antihistamine drugs
prescribed are to control allergic symptoms. In any
case, the immunomodulatory effects of these drugs are
important. Previous studies and our study have shown
that these drugs are able to modulate allergic reaction;
therefore INCs and antihistamine as first-line for
treatment of patients are suggested. The assessment of
cytokine profiles of PBMCs of AR patients several
Effect of Intranasal Corticosteroid and Oral Antihistamine Treatment on Allergic Rhinitis
Vol. 9, No. 4, December 2010 IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /229
times during the course of prescribed drugs is limitation
of this study. The results obtained in this experimental
study could be used for further investigations to explore
the involvement of cytokines effects and potency of
INC and antihistamine compounds in protection of
allergic reactions.

ACKNOWLEDGEMENTS

We gratefully thanks to Mr. Behnam Asadifar, Mr.
Reza Ghelman, Mr. Farhad J alali, Miss. Ensie
Akbarzadeh, Miss. Roghaye Zolghadr and the Fellows
of allergy ward, especially Dr. Behrang Taghvaee.

REFERENCES

1. Sibbald B, Rink E. Epidemiology of seasonal and
perennial rhinitis: clinical presentation and medical
history. Thorax 1991; 46(12):895901.
2. Mohammadi K, Gharagozlou M, Movahedi M. A single
center study of clinical and paraclinical aspects in Iranian
patients with allergic rhinitis. Iran J Allergy Asthma
Immunol 2008; 7(3):163-7.
3. Mousavi T, Movahedi M, Asadi N, Farhoodi A, Moin M.
The comparison of conventional and W.H.O methods for
protein determination of Chenopodium album allergic
extract. Iran J Allergy Asthma and Immunol 2003,
2(2):106-9.
4. Mousavi T, Asadi N, Tebyanian M. Study of
Chenopodium Album Allergenic Extract to Induce
Allergic Asthma in Murine Model. IJ I 2005; 2(3): 166-
71.
5. Larche M, Robinson DS, Kay AB. The role of T
lymphocytes in the pathogenesis of asthma. J Allergy
Clin Immunol 2003; 111(3):45063.
6. Hershey GK. IL-13 receptors and signaling pathways: an
evolving web. J Allergy Clin Immunol 2003; 111(4):677
90.
7. Bousquet J , Khaltaev N, Cruz AA, Denburg J , Fokkens
WJ , Togias A, et al. Allergic rhinitis and its impact on
asthma. ARIA. In collaboration with the World Health
Organization. J Allergy Clin Immunol 2001; 108:1315.
8. Masuyama K, Till SJ , J acobson MR, Kamil A, Cameron
L, J uliusson S, et al. Nasal eosinophilia and IL-5 mRNA
expression in seasonal allergic rhinitis induced by natural
allergen exposure: effects of topical corticosteroid. J
Allergy Clin Immunol 1998; 102(4 pt 1):61017.
9. Linden M, Svensson C, Andersson E, Andersson M,
Greiff L, Persson CG. Immediate effect of topical
budesonide on allergen challenge-induced nasal mucosal
uid levels of granulocyte-macrophage colony-
stimulating factor and interleukin-5. Am J Respir Crit
Care Med 2000; 162(5):17058.
10. SimTC, Reece LM, Hilsmeier KA, Grant J A, Alam R.
Secretion of chemokines and other cytokines in allergen-
induced nasal responses: inhibition by topical steroid
treatment. Am J Respir Crit Care Med 1995; 152(3):927
33.
11. Ciprandi G, Tosca MA, Passalacqua G, Canonica GW.
Intranasal mometasone furoate reduces late-phase
inammation after allergen challenge. Ann Allergy
Asthma Immunol 2001; 86(4):4338.
12. Greiff L, Petersen H, Mattsson E, Andersson M, Erjeflt
J S, Linden M, et al. Mucosal output of eotaxin allergic
rhinitis and its attenuation by topical glucocorticosteroid
in treatment. Clin Exp Allergy 2001; 31(8):13217.
13. Domly PD, Bousquet J , Romano A. In Vivo Methods for
the Study of Allergy. In: Adkinson NF, Bochner BS,
editors. Middleton's Allergy Principles & Practice. USA:
Elsevier, 2008: 1267-78.
14. Varney VA, J acobson MR, Sudderick RM, Robinson DS,
Irani A, Schwartz LB, et al. Immunohistology of the nasal
mucosa following allergen-induced rhinitis: identification
of activated T lymphocytes, eosinophils, and neutrophils.
AmRev Respir Dis 1992; 146(1):1706.
15. Durham SR, Ying S, Varney VA, J acobson MR,
Sudderick RM, Mackay IS, et al. Cytokine messenger
RNA expression for IL-3, IL-4, IL-5, and
granulocyte/macrophage-colony-stimulating factor in the
nasal mucosa after local allergen provocation:
relationship to tissue eosinophilia. J Immunol 1992;
148(8):23904.
16. Bufford J D, Gern J E. The hygiene hypothesis revisited.
Immunol Allergy Clin North Am2005; 25(2):24762.
17. Akdis M, Verhagen J , Taylor A, Karamloo F,
Karagiannidis C, Crameri R, et al. Immune responses in
normal and allergic individuals are characterized by a ne
balance between allergen-specic T regulatory 1 and T
helper 2 cells. J Exp Med 2004; 199: 156775.
18. Ostroukhova M, Ray A. CD25+T cells and regulation of
allergen-induced responses. Curr Allergy Asthma Rep
2005; 5(1):3541.
19. Rudin A, Macaubas C, Wee C, Holt BJ , Slya PD, Holt
PG. "Bystander" amplification of PBMC cytokine
responses to seasonal allergen in polysensitive atopic
children. Allergy 2001; 56 (11): 1042-8.
20. Heaton T, Rowe J , Turner S, Aalberse RC, de Klerk N,
Suriyaarachchi D, et al. An immunoepidemiological
Sh. Farrokhi, et al.
230/ IRANIAN J OURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Vol. 9, No. 4, December 2010
approach to asthma: identi cation of in -vitro T-cell
response patterns associated with different wheezing
phenotypes in children. Lancet 2005; 365(9454):1429.
21. Rak S, J acobson MR, Sudderick RM, Masuyama K,
J uliusson S, Kay AB, et al. Influence of prolonged
treatment with topical corticosteroid (fluticasone
propionate) on early and late phase nasal responses and
cellular infiltration in the nasal mucosa after allergen
challenge. Clin Exp Allergy 1994; 24(10):9309.
22. Masuyama K, J acobson, Rak S, Meng Q, Sudderick RM,
Kay AB, et al. Topical glucocorticosteroid (fluticasone
propionate) inhibits cells expressing cytokine mRNA for
interleukin-4 in the nasal mucosa in allergen-induced
rhinitis. Immunology 1994; 82(2):1929.
23. Linden M, Svensson C, Andersson E, Andersson M,
Greiff L, Persson CG. Immediate effect of topical
budesonide on allergen challenge-induced nasal mucosal
uid levels of granulocyte- macrophage colony-
stimulating factor and interleukin-5. Am J Respir Crit
Care Med 2000; 162(5): 17058.
24. Ghaffar O, Laberge S, J acobson MR, Lowhagen O, Rak
S, DurhamSR, et al. IL-13 mRNA and immunoreactivity
in allergen-induced rhinitis: comparison with IL-4
expression and modulation by topical glucocorticoid
therapy. AmJ Respir Cell Mol Biol 1997; 17(1):1724.
25. Wolk K, Kunz S, Asadullah K, Sabat R. Cutting edge:
immune cells as sources and targets of the IL-10 family
members? J Immunol 2002; 168(11):5397402.
26. SimTC, Grant J A, Kimberly A, Hilsmeier H, Fukuda Y,
Alam R. Proinflammatory cytokines in nasal secretions of
allergic subjects after antigen challenge. Am J Respir Crit
Care Med 1994; 149(2 pt 1):33944.


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